Ultra-trace small RNA (Ribonucleic Acid) sequencing method

Abstract

The invention relates to an ultramicro small RNA (Ribonucleic Acid) sequencing method, which adopts a tubular reaction, carries out RNA terminal repair and AlKB demodification enzyme activity reaction by expanding a system step by step, and comprises cell lysis, RNA terminal repair, 3'adapter connection, AlKB demodification enzyme activity reaction, RNA purification, RT (Reverse Transcription) annealing, 5 'adapter connection, cDNA (Complementary Deoxyribose Nucleic Acid) reverse transcription, library amplification and NGS (Next Generation System). According to the present invention, the improvement is performed on the basis of the existing PANDORA-Seq technology, the efficient and accurate detection of the trace cell small RNA is achieved by optimizing the reaction conditions, and the UltraTra-PANDORA-Seq method is successfully established. According to the method, the technical problem of the traditional method in trace cell small RNA detection is solved, and a feasible and reliable analysis means is provided for deep research on the functional mechanism of the small RNA in a living body. The development of the Ultra-PANDORA-Seq technology provides important scientific basis and technical support for exploration of intergenerational genetic mechanisms of metabolic diseases, early clinical diagnosis, development of small RNA drugs and clinical intervention.

Description

Technical Field

[0001] The invention relates to the field of biotechnology, in particular to an ultra-trace small RNA sequencing method. Background Art

[0002] Existing trace small RNA sequencing technology can measure the dynamic changes of small RNAs such as miRNA and piRNA in trace cells [1]. However, after the development of PANDORA-Seq technology by the present invention team in 2021, it was found that in addition to miRNA and piRNA, there are also a large number of small RNA types such as rsRNA and tsRNA in cells [2]. Existing small RNA sequencing technology is still unable to accurately analyze these small RNAs. Therefore, it is urgent to establish a new type of ultra-trace small RNA sequencing technology to more accurately reveal the true change characteristics of small RNAs in trace cells (such as embryos).

[0003] Current sequencing technologies have certain limitations: on the one hand, some technologies can detect trace amounts of small RNA, but fail to fully present the complete expression change profile of small RNA; on the other hand, PANDORA-Seq technology can provide a relatively comprehensive RNA change profile, but it is still insufficient in the detection accuracy of trace amounts of small RNA. Therefore, there is an urgent need for an ultra-trace, high-precision small RNA sequencing method to more accurately depict the true change characteristics of small RNA in cells. Summary of the invention

[0004] The purpose of the present invention is to provide an ultra-trace small RNA sequencing technology to address the deficiencies in the prior art and to achieve accurate detection of the expression pattern of trace cellular small RNA.

[0005] In a first aspect, the present invention provides an ultra-trace small RNA sequencing method, which adopts a one-tube reaction and performs RNA end repair and AlKB demodification enzyme activity reaction through a step-by-step expansion system, including cell lysis, RNA end repair, 3' adapter ligation, AlKB demodification enzyme activity reaction, RNA purification, RT annealing, 5' adapter ligation, cDNA reverse transcription, library amplification and NGS.

[0006] As a preferred example, the RNA end repair refers to T4PNK treatment.

[0007] As a preferred example, the AlKB demodification enzymatic activity reaction utilizes intracellular ferrous ions to assist the enzymatic activity reaction of AlKB to remove multiple RNA modifications on small RNAs.

[0008] As a preferred example, the 3' linker ligation refers to first performing 3' terminal linker ligation and then performing AlKB enzyme demodification reaction.

[0009] As a preferred example, the sequencing method directly uses cell lysate to perform small RNA library construction process.

[0010] As a preferred example, the sequencing method can construct the library using a small amount of cells.

[0011] As a preferred example, the trace cells are at least 10 cells and 2 fertilized eggs.

[0012] The present invention has the advantages that:

[0013] The present invention has made improvements on the existing PANDORA-Seq technology, and has achieved efficient and accurate detection of trace cellular small RNAs by optimizing the reaction conditions, and has successfully established the Ultra-PANDORA-Seq method. The present invention overcomes the technical difficulties of traditional methods in detecting trace cellular small RNAs, and provides a feasible and reliable analytical method for in-depth research on the functional mechanisms of small RNAs in organisms. In the study of intergenerational inheritance of metabolic diseases, small RNAs, as an important medium connecting parents and offspring, have always been a research difficulty in the field of metabolism. The development of Ultra-PANDORA-Seq technology provides important scientific basis and technical support for the exploration of intergenerational inheritance mechanisms of metabolic diseases, early clinical diagnosis, small RNA drug development and clinical intervention.

[0014] PANDORA-Seq sequencing method: This method requires at least 100,000 cells to conduct the experiment and cannot be performed on trace cell samples.

[0015] The sequencing method of Ultra-PANDORA-Seq of the present invention: directly using cell lysate for small RNA library construction process, reducing the total RNA extraction and small RNA fragment separation steps. During the experiment, efficient library construction can be performed using a trace amount of cells (as low as 10 cells and 2 fertilized eggs).

[0016] PANDORA-Seq sequencing method: This method is cumbersome and requires the extraction of total cellular RNA first, followed by the recovery of small RNA fragments (15-50 nt), and then the subsequent library construction process. During this period, the extraction of total cellular RNA and the recovery of small RNA fragments may cause RNA degradation, resulting in sequencing preference.

[0017] The sequencing method of Ultra-PANDORA-Seq of the present invention adopts a one-tube reaction and performs RNA end repair (T4PNK treatment) and AlKB demodification enzyme activity reaction by step-by-step expansion of the system. The method eliminates the RNA recovery step after T4PNK treatment of cells, avoiding RNA loss caused by RNA recovery and unexpected RNA degradation problems.

[0018] PANDORA-Seq sequencing method: The efficiency of gel recovery of small RNA is relatively low, which will lead to a disproportionate loss of small RNA fragments before small RNA library construction, causing sequencing bias.

[0019] The sequencing method of Ultra-PANDORA-Seq of the present invention utilizes ferrous ions in cells to assist the enzymatic activity reaction of AlKB, thereby effectively removing various RNA modifications on small RNAs. This optimization step avoids the RNA degradation problem caused by the additional addition of ferrous ions, and effectively solves the endogenous contamination problem of small RNAs, especially tsRNA and rsRNA.

[0020] PANDORA-Seq sequencing method: After the small RNA undergoes terminal modification reaction (T4PNK treatment), the RNA needs to be re-extracted and recovered before entering the subsequent AlKB enzyme de-modification reaction. The additional RNA extraction step in the middle is complicated and may further cause the loss of small RNA.

[0021] The sequencing method of Ultra-PANDORA-Seq of the present invention adopts the method of first performing 3' end adapter connection and then performing AlKB enzyme demodification reaction. This step optimization effectively solves the problem of small RNA contamination introduced by AlKB and the problem of RNA degradation caused by AlKB treatment. It comprehensively solves the problem of exogenous contamination caused by AlKB enzyme and endogenous contamination caused by cellular RNA degradation in the sequencing process of trace samples. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 .Ultra-PANDORA-Seq experimental flow chart.

[0023] Figure 2 .Data analysis diagram of two sequencing methods, Ultra-PANDORA-seq and PANDORA-seq. The results show that the sequencing results of small RNA of ultra-trace cells by the Ultra-PANDORA-seq technology of the present invention and the PANDORA-seq technology for a large number of samples have achieved highly consistent sequencing results, achieving accurate analysis of small RNA of trace samples. DETAILED DESCRIPTION

[0024] The present invention will be further described below in conjunction with specific embodiments. It should be understood that these embodiments are only used to illustrate the present invention and are not intended to limit the scope of the present invention. In addition, it should be understood that after reading the content of the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms fall within the scope limited by the appended claims of the application equally.

[0025] Example 1

[0026] The Ultra-PANDORA-Seq flow chart of the present invention is shown in Figure 1

[0027] (1) Specific experimental materials and methods:

[0028] 1) Lyse cells

[0029] Place 100 cells into a 0.2 mL PCR tube containing 4 μL of cell lysis buffer.

[0030] RNase-free H2O (purchased from Invitrogen, catalog number: 10977015) and 4U of RNase inhibitor (purchased from Vazyme, catalog number: RL301). Carefully resuspend the cells with a pipette and place them on

[0031] Freeze in -80° refrigerator for 30 min, or freeze in liquid nitrogen for 5 min. Take the sample out of the refrigerator, thaw on ice, centrifuge at low speed, and incubate at 72° for 3 min. After the reaction is completed, place the sample on ice immediately and proceed to the next step.

[0032] 2) Small RNA end repair

[0033] T4PNK was used for RNA end repair. T4PNK was purchased from NEB, catalog number: M0201L. 12URNase inhibitor (purchased from Vazyme, catalog number: RL301) was added during the reaction. The following reaction system was configured:

[0034]

[0035] Premix the reaction reagents and add 2 μL to the reaction system in the previous step. Mix gently and incubate at 37°C for 20 minutes. After the reaction is completed, place on ice for the next step of linker ligation reaction.

[0036] 3) 3' adapter ligation

[0037] After the previous step, perform 3' adapter ligation, referring to the VAHTS Small RNA Library Prep Kit for Illumina V2 kit (purchased from Vazyme, catalog number: NR811). First, dilute the adapter at 1:39, then take 1μL of the adapter and mix it with the previous step reaction solution, place it at 70℃, denature for 2min, and immediately place it on ice for 2min after the reaction. Add RL3 buffer and RL3-enzyme in sequence, the system is as follows, the reaction conditions are 25℃ reaction for 1h, 75℃ denaturation for 5min.

[0038]

[0039] 4) AlKB removal modification

[0040] 10 minutes before the end of the previous step, the AlKB removal modification reaction system was prepared, which contained 50mM HEPES (pH 8.0) (purchased from Gibco, Catalog No.: 1563008), 1mM α-ketoglutaric acid (purchased from Sigma-Aldrich, Catalog No.: K1128-25G), 2mM sodium ascorbate, 1ng / mL AlKB and D135S AlKB, and 40U RNase inhibitor. The above reagents were premixed, and after the reaction was completed, 30μL of the premix was added to the reaction solution of the previous step and gently mixed with a pipette tip.

[0041] The specific reaction system is shown in the following table, and the reaction conditions are incubation at 37°C for 30 min.

[0042]

[0043] 5) RNA purification

[0044] 10 minutes before the end of the previous step, prepare a 2mL Phase Lock Gel tube (purchased from TIANGEN, catalog number: WM5-2302820), add 150μL TRIzol reagent (purchased from Invitrogen, catalog number: 15596018) in advance, and after the previous step, take out the PCR tube, centrifuge briefly and quickly transfer the liquid to the Phase Lock Gel tube, vortex the liquid thoroughly, and incubate it at room temperature for 5 minutes.

[0045] min, add 40 μL chloroform, vortex quickly for 15 seconds, incubate at room temperature for 7 minutes, and then centrifuge at 12000g (4°C) for 15 minutes. Collect the upper aqueous phase and transfer it to a new 1.5mL EP tube, then add 100 μL isopropanol and slowly invert to mix, then add 2.5 μL glycogen (purchased from Invitrogen, catalog number:

[0046] AM9510), gently mix and place in -80℃ refrigerator to precipitate for 30min or overnight. After precipitation, centrifuge at 13000rpm (4℃) for 30min. After removing the supernatant, wash the precipitate with 1mL75% ethanol and centrifuge at 13000rpm (4℃) for 5min. Remove the supernatant again and place in air to dry for 5min.

[0047] 6) RT annealing

[0048] Add 6.75μL RNase-free H2O to the 1.5mL EP tube dried in the previous step, gently mix the liquid, and transfer it to a new 0.2mL PCR tube for RT annealing reaction. Dilute RTPrimer at 1:39, add RTPrimer and RL3 buffer to the reaction solution in the previous step in sequence, and gently mix with a pipette tip. The reaction system is as follows: the reaction conditions are 75℃ denaturation for 5min, 37℃ reaction for 15min, and 25℃ incubation for 15min.

[0049]

[0050] 7) 5' adapter ligation

[0051] After the reaction in the previous step, the 5' adapter was immediately connected. The adapter was RNA and needed to be diluted at 1:39 and denatured at 70℃ for 2 minutes. After the reaction, it was placed on ice for later use. The adapter connection contained RL5 buffer and RL5 enzyme. The reaction system was as follows, and the reaction conditions were incubation at 25℃ for 90 minutes.

[0052]

[0053] 8) cDNA reverse transcription

[0054] After the previous step, cDNA reverse transcription was performed immediately. The buffer required for reverse transcription was thawed on ice. The reaction contained RT buffer and RT enzyme. The following reaction system was configured. The reaction conditions were 50°C for 60 min and 80°C for denaturation for 5 min.

[0055]

[0056] 9) Library amplification

[0057] After the previous step, you can directly perform PCR library amplification, or store the cDNA in a -80℃ refrigerator and then perform library amplification (-80℃ refrigerator can be stored for at least 1 week). The reagents required for library amplification include Amplificationmix 3, Index Primer and Universal Primer. The obtained cDNA is configured according to the following system:

[0058]

[0059] The reaction conditions are as follows

[0060]

[0061] 9) NGS

[0062] The PCR product was purified using DNA Clean Beads (purchased from Vazyme, catalog number: N411) according to the instructions. After purification, the product was sent to a sequencing company for quality inspection and sequencing.

[0063] (2) Analysis of database construction results

[0064] The PANDORA-Seq method for large numbers of cells was compared with the sequencing results of the Ultra-PANDORA-Seq method for small numbers of cells that we established. The results showed that the ultra-micro-cell sequencing method we established can achieve small-scale micro-cell detection while achieving a similar small RNA expression pattern as that of large-scale cell detection. Figure 2 As shown on the left, different sequencing methods have detected that rsRNA accounts for the largest proportion of small RNA, followed by tsRNA and other. From the sequence distribution on the right, we can see that the sequence distributions measured by large and small methods are roughly the same. Figure 2 The scatter plot correlation analysis below further shows that the data obtained by the two methods are highly similar. The above results show that the ultra-trace Ultra-PANDORA-Seq we established can achieve high-precision detection of dynamic changes of small RNA in trace cells.

[0065] References

[0066] [1] Yang Q, Lin J, LiuM, LiR, TianB, ZhangX, XuB, LiuM, ZhangX, Li Y, Shi H, WuL. Highly sensitive sequencing reveals dynamic modifications and activities of small RNAs in mouse oocytes and early embryos. Sci Adv. 2016 Jun 10; 2(6): e1501482.

[0067] [2]Shi J, Zhang Y, Tan D, et al. PANDORA-seq expands the repertoire ofregμLatory small RNAs by overcoming RNA modifications. Nat Cell Biol. 2021Apr; 23(4):424-436.

[0068] The above is only a preferred embodiment of the present invention. It should be pointed out that ordinary technicians in this technical field can make several improvements and supplements without departing from the method of the present invention. These improvements and supplements should also be regarded as the scope of protection of the present invention.

Claims

1. A method for sequencing ultra-trace small RNAs, characterized in that: The sequencing method adopts a one-tube reaction and performs RNA end repair and AlKB demodification enzyme activity reaction through a step-by-step expansion system, including cell lysis, RNA end repair, 3' adapter ligation, AlKB demodification enzyme activity reaction, RNA purification, RT annealing, 5' adapter ligation, cDNA reverse transcription, library amplification and NGS.

2. The sequencing method according to claim 1, characterized in that The RNA end repair refers to T4PNK treatment.

3. The sequencing method according to claim 1, characterized in that The AlKB demodification enzymatic activity reaction utilizes intracellular ferrous ions to assist the enzymatic activity reaction of AlKB to remove various RNA modifications on small RNAs.

4. The sequencing method according to claim 1, characterized in that The 3' linker connection refers to firstly performing 3' terminal linker connection and then performing AlKB enzyme demodification reaction.

5. The sequencing method according to claim 1, characterized in that The sequencing method directly uses cell lysate to perform small RNA library construction process.

6. The sequencing method according to claim 1, characterized in that The sequencing method can construct a library using a small amount of cells.

7. The sequencing method according to claim 6, characterized in that Microcells are at least 10 cells and 2 fertilized eggs.

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